Summary

Despite the presence of more than 400 genes that encode receptor-like
kinases (RLKs) in the Arabidopsis thaliana genome, very little is
known about the range of biological processes that they control, or the
mechanisms by which they function. This review focuses on the most recent
findings from studies of several leucine-rich-repeat (LRR) class RLKs in
A. thaliana, and their implications for our understanding of plant
receptor function and signaling. We compare the biological functions of plant
and animal LRR-containing receptors, and the potential commonalities in the
signaling mechanisms employed.

Introduction

One of the fundamental mechanisms by which cells communicate in
multicellular organisms is through the secretion of ligands that then bind to
cell surface receptors possessing protein kinase catalytic activities. Given
that plant cells are separated from one another by cell walls, it was assumed
for decades that most cell-to-cell communication would occur via the
cytoplasmic bridges called plasmodesmata. However, in 1990, Walker and Zhang
identified the first plant receptor-like kinase (RLK) in maize
(Walker and Zhang, 1990) and,
since then, many RLKs have been identified from the flowering plant
Arabidopsis thaliana and other plant species. These findings
demonstrate that, like other eucaryotes, plant cells are able to perceive
external signals at the plasma membrane.

An extensive phylogenic analysis of the RLKs in A. thaliana has
revealed that more than 400 genes encode putative plant receptor kinases
(PRKs) (reviewed by Tichtinsky et al.,
2003; Shiu and Bleecker,
2001a; Shiu and Bleecker,
2001b), defined as proteins that contain an extracellular domain,
a single-pass transmembrane domain and a cytoplasmic serine/threonine
(ser/thr) protein kinase domain (Shiu and
Bleecker, 2001a; Shiu and
Bleecker, 2001b). The PRKs can be classified on the basis of their
extracellular domains (Shiu and Bleecker,
2002). Leucine-rich repeat (LRR)-containing PRKs represent the
largest group of PRKs in the A. thaliana genome, with 216 members.
These represent 13 subfamilies (LRR I to XIII), which can be classified
according to the organization of the LRRs in the extracellular domain
(Shiu and Bleecker, 2002).
Plant LRR-RLKs possess a functional cytoplasmic kinase domain, and all of the
plant LRR-RLKs analyzed to date possess ser/thr kinase activity. The
phenotypes associated with mutations in various LRR-PRKs show that they play
roles in diverse processes during growth and development
(Table 1A).

Function of LRR-RLKs in plants and LRR-containing receptors in
animals

In animals, numerous studies have reported functions for LRR-containing
receptors in neuronal development, pattern formation, differentiation and
growth of gonads and thyroid glands, and antifungal responses
(Table 1B). These receptors
contain LRRs in the extracellular portion of the protein, and either possess a
transmembrane domain(s) or are anchored to the plasma membrane by a
glycosylphosphatidylinositol (GPI) linkage. However, unlike plant LRR-RLKs,
animal LRR-containing receptors do not contain a cytoplasmic kinase domain,
but instead are thought to transduce their signal through the activation of
co-receptors. Nevertheless, the LRR motifs found in the extracellular part of
these receptors are alike in plants and animals, indicating that they could
share similar mechanisms of ligand binding and activation.

This review will focus on the developmental processes that are affected by
mutations in LRR-containing receptors, describing what we know about the
ligands, the receptors and the pathways that are activated during some
signaling pathways in plants. Out of the 216 LRR-RLKs in A. thaliana,
only 10 or so have known functions, and only four have been extensively
studied. Presented in Table 1
are many of the LRR receptors of plants, and of other organisms, for which
functions have been attributed. Recent studies, in plants and animals, have
led to a better understanding of a number of signaling pathways that involve
LRR-containing receptors, and a comparison of the recent advances gives us
some insights into the conserved and divergent signaling mechanisms within
plants, and between plants and animals.

The CLAVATA pathway

The development of higher plants is largely postembryonic, and plant
embryos contain few of the organs found in the adult plant. Plant embryos are
simple in structure (Fig. 1).
The apical end of the embryo contains a shoot meristem, which produces the
above-ground organs and tissue of the plant, namely the stems, the leaves and
the flowers. The basal end of the embryo contains a root meristem, which gives
rise to the root system. Plant organs are formed from shoot and root meristems
during post-embryonic development. Cytohistological examination of shoot
meristems has revealed that the shoot apex is partitioned into radial domains
(Fig. 2D)
(Steeves and Sussex, 1989).
This organization defines at least three functionally distinct zones of the
shoot meristem: the central zone (CZ), the peripheral zone (PZ) and the rib
zone (RZ). The CZ is composed of stem cells with low mitotic activity. This CZ
is surrounded at the meristem flanks by the PZ
(Fig. 2D), where progeny of the
stem cells divide more rapidly than those at the center and are incorporated
into organ primordia. Underneath these two zones, the RZ gives rise to the
internal part of the stems. As the size and shape of the shoot meristem are
defined early in embryogenesis and remain relatively constant during normal
development, the meristem has to maintain a tight balance between the
proliferation of stem cells at the CZ and the targeting of these cells towards
differentiation at the periphery. Very similar and evolutionaily related zones
to those found in the shoot meristem are found within flower meristems during
the initiation of flower organs. Within the last few years, genetic analyses,
along with gene expression and biochemical data, have uncovered the importance
of cell-cell interactions, and the involvement of an LRR-RLK, in maintaining
the size homeostasis of meristems.

Life cycle of Arabidopsis thaliana. The mature plant possesses
primary and secondary roots, rosette and cauline leaves, and inflorescences.
Flowers are composed of sepals, petals, stamens (male reproductive organs) and
carpels (female reproductive organs). After pollination, the fertilized egg
develops into an embryo inside the silique. The embryo possesses two
meristems, a shoot meristem (SAM) and a root meristem (RAM), where new organs
and tissues are initiated during post-embryonic growth. Seeds germinate and
give rise to seedlings composed of the embryonically-formed hypocotyl and
cotyledons. Further organogenesis depends on the action of shoot and root
meristems.

The shoot meristem. Scanning electron microscopy of a wild-type shoot
meristem (A) reveals little morphological differentiation, except for the
distinct organ primorida (op), whereas molecular and detailed histological
studies have revealed important subdomains (PZ, peripheral zone; CZ, central
zone; RZ, rib zone) of the shoot meristem (D). The flower meristem gives rise
to a regular number of floral organs and organ types (B), eventually forming
the fruit (also called the silique; C). In plants carrying a mutation in one
of the CLV genes, the shoot meristem can be massively enlarged (E),
as a result of expansion of the population of stem cells (H). clv
flower meristems also accumulate stem cells, resulting in the formation of
additional organs of each type (F), and leading to distorted, club-shaped
fruits (G). (I) A model for CLV signaling. The CLV3 protein (blue) is secreted
from the stem cells and diffuses to the underlying cell layers, where CLV1
(red) is expressed. There, CLV1 binds to CLV3 and activates signaling to
repress WUS expression, as well as to sequester CLV3 to prevent it
from diffusing to the WUS-expressing region. CLV2 (green) is a
putative co-receptor with CLV1. Scale bar in A and E: 50 μm.

In A. thaliana, loss-of-function mutations in any of the three
CLAVATA (CLV) genes (CLV1, CLV2 and CLV3)
cause an ectopic accumulation of stem cells in the CZ, and a progressive
enlargement of the shoot meristem (Fig.
2). Very similar defects are found within clv flowers, in
which an early accumulation of stem cells leads to additional organs formed of
each type and the continued proliferation of the flower beyond the normally
terminal carpels. These clv mutant phenotypes result in club-shaped
(clavatus in Latin) fruits (Fig.
2). Although mutations of all three genes lead to similar
phenotypes, the clv1 null mutant has a subtle phenotype, whereas the
phenotype of the clv3 null mutant is more severe. The CLV
loci encode signal transduction components. CLV1 is an LRR-RLK, which
possesses an extracellular domain containing 21 LRRs, a transmembrane domain
and a functional cytoplasmic ser/thr kinase domain
(Clark et al., 1997;
Williams et al., 1997;
Stone et al., 1998). It is
predicted that CLV1 is targeted to the plasma membrane, but its subcellular
localization has not yet been demonstrated. CLV2 is a receptor-like protein,
composed of 21 LRRs in the predicted extracellular part of the receptor (which
contains an `island' of non-LRR sequence between the 17th and the 18th
repeats) and a short predicted cytoplasmic tail that has no known sequence
motifs (Jeong et al., 1999;
Kayes and Clark, 1998). CLV3
is a small 96-amino acid polypeptide that may act as a ligand for the CLV1
and/or CLV2 receptors (Fletcher et al.,
1999; Rojo et al.,
2002).

Molecular genetic studies have indicated that the primary function of
proteins encoded by the CLV loci is to restrict the expression domain
of a stem-cell promoting factor called WUSCHEL (WUS), which
encodes a homeodomain transcription factor
(Laux et al., 1996;
Mayer et al., 1998).
WUS is necessary for stem cell specification; wus mutants
repeatedly form shoots and flowers that lack stem cells, and, as a
consequence, lack extended organogenesis
(Laux et al., 1996). The
wus mutation is fully epistatic to clv mutations, indicating
that WUS acts downstream of the CLV1 pathway. WUS is expressed in a
small group of cells situated below the CZ of the shoot and flower meristems,
called the organizing center (OC) (Mayer
et al., 1998). The mechanism by which WUS expression in
the underlying OC leads to stem cell specification in overlying CZ cells is
unclear. In shoot apices, WUS overexpression is sufficient to induce
stem cell identity, and also CLV3 gene expression in adjacent cells
(Brand et al., 2000;
Schoof et al., 2000). The
induction of CLV3 by WUS expression forms a putative
feedback loop that regulates the size of the stem cell population in the shoot
meristem, in which CLV3 restricts expression of WUS and WUS
induces the expression of CLV3
(Schoof et al., 2000).

Several recent studies have revealed key aspects of CLV signaling. An
elegant study carried out by Fletcher and coworkers has shown that for CLV3 to
function, it must be secreted into the extracellular space within the meristem
(Rojo et al., 2002). They
assessed the importance of the signal peptide and the secretion of CLV3 for
the proper function of CLV3, using CLV3-GFP fusion cDNAs
driven by the constitutive viral 35S promoter. These constructs induced the
constitutive production of CLV3-GFP fusion proteins. In clv3
transgenic plants possessing a version of this construct in which the putative
signal peptide of the CLV3-GFP fusion protein is deleted, the protein is
localized in the cell cytoplasm, and no rescue of the clv3 phenotype
is observed. However, when a wild-type full-length CLV3-GFP fusion
cDNA is used, the protein is detected outside of the cells in the apoplastic
space, and it is able to suppress the accumulation of meristematic cells in
the clv3 mutant plants. Targeting CLV3, through the secretory system,
to the vacuole by use of a vacuolar sorting signal from barley lectin also
blocked CLV3 function (Rojo et al.,
2002). These experiments demonstrate that CLV3 must be secreted
into the extracellular space within the meristem in order to function.

Experiments performed by Lenhard and Laux have gone further to determine
whether or not CLV3 proteins can move within the shoot meristem
(Lenhard and Laux, 2003). They
observed that when a CLV3-GFP fusion construct was expressed under
the control of the endogenous CLV3 promoter and transformed into
clv3 mutant plants, CLV3-GFP could diffuse within the shoot meristem
to positions several cells away from the stem cell that secreted it, and could
suppress the clv3 mutant phenotype. Furthermore, expression of CLV3
protein from an epidermis-specific promoter not only suppresses the mutant
phenotype of clv3 plants, but also results in a wus-like
phenotype with early termination of the meristem. This indicates that the CLV3
protein can diffuse from the epidermal cells to the center of the meristem
where WUS expression is normally maintained. This long-range effect
was not observed in a clv1 mutant background, indicating that the
activity of CLV3, synthesised under the control of an epidermis-specific
promoter, is dependent on functional CLV1. To test whether the CLV1 receptor
was able to sequester the CLV3 protein, expression of both CLV1 and
CLV3-GFP was driven by an epidermis-specific promoter. In this
experiment, strong CLV3-GFP fluorescence was restricted to the epidermis,
which was in contrast to the more diffuse fluorescence observed when CLV3-GFP
was expressed alone. This showed that CLV1 can restrict the movement of
CLV3-GFP, and indicated that in wild-type plants, CLV3 does not reach the OC
because of its sequestration by CLV1 outside of this region. The implications
of these studies are that CLV1 both responds to the CLV3 signal and limits its
diffusion, making CLV1 both a positive mediator of the CLV3 signal, and a key
factor to limit where CLV3 acts.

The next experiment was designed to test whether overexpression of CLV3
from the stem cells would decrease the WUS expression domain and the
size of the meristem. Indeed, the expression of five copies of CLV3
under its own promoter in otherwise wild-type plants reduced the meristem size
by more than 20% compared with the control, and correlated with a smaller
WUS-expression domain. The small difference in the WUS
expression domain measured between wild-type and transgenic meristems
indicated that the proposed regulatory feedback loop between WUS and
CLV3 is able to prevent the meristem termination in this experiment.
Furthermore, to confirm that the reduction in meristem size was due to an
excess of CLV3 protein secreted by the stem cells, the researchers also
expressed CLV1 under the CLV3 promoter in the transgenic
plants expressing five copies of CLV3. CLV1 was expected to
immediately sequester CLV3, preventing it from functioning and, indeed, in
this case, the meristem size was increased by 20%
(Lenhard and Laux, 2003).
These experiments showed that CLV3 proteins, although produced in the stem
cells, can move away to neighboring cells, where they activate the CLV1
pathway to repress WUS expression.

Another recent study strongly indicates the presence of an additional
receptor kinase(s) that functions redundantly with CLV1 during the regulation
of meristem development (Diévart et
al., 2003). Isolation of insertional alleles within clv1
mutant plants revealed that clv1 null alleles display fairly weak
phenotypes. All of the strong and intermediate clv1 alleles
characterized to date contain missense mutations, and these alleles are
therefore most likely to be dominant negative. Consistent with this
hypothesis, co-suppression of the intermediate clv1-1 allele in
transgenic plants partially rescued the mutant phenotype, such that the
co-suppressed plants appears phenotypically similar to clv1 null
mutants, which are weak in phenotype. Thus, suppressing expression of the
dominant-negative clv1-1 isoforms reduced the severity of the mutant
phenotype. Because the clv3 null allele exhibits the strongest
phenotype among the clv alleles, and because clv1 null
alleles are rather weak in phenotype, it is likely that there is an additional
receptor kinase(s) capable of relaying the CLV3 signal in the absence of CLV1.
This redundant receptor could be inactivated by the presence of clv1
dominant-negative isoforms, indicating receptor multimerization during the
activation of CLV1 signaling. Interestingly, clv1 dominant-negative
alleles exhibit major differences from dominant-negative alleles characterized
among animal receptor kinases, including the ability of missense mutations in
the extracellular domain to act in a dominant-negative manner.

Both genetic and biochemical studies have been used to try to determine the
components of the signaling pathway that function downstream of CLV1. Many
animal receptor kinases associate with small GTPases of the Ras superfamily to
relay signal to downstream targets such as a mitogen-activated protein kinase
(MAPK) pathway. Although there is no evidence for a Ras ortholog in plants,
there is a plant-specific family of small Rho-related GTPases, termed Rop
(Li et al., 1998;
Winge et al., 1997).
Co-immunoprecipitation experiments have revealed that CLV1 may be associated
in a complex with one Rop protein
(Trotochaud et al., 1999).
Although the detected protein is antigenically-related to a known Rop protein
in A. thaliana, it remains to be determined whether the detected
protein is indeed a member of the Rop family and whether the binding to CLV1
is direct or mediated by linker proteins.

The second protein phosphatase, POL, also regulates the CLV1 pathway
(Yu et al., 2003;
Yu et al., 2000). POL
encodes a predicted nuclear-localized PP2C with a putative N-terminal
regulatory domain, and, on the basis of phylogenetic analyses, POL represents
a new subclass of plant PP2Cs (Yu et al.,
2003). POL protein synthesized in E. coli displays
phosphatase catalytic activity, but only if an amino-terminal domain is
removed (Yu et al., 2003).
The ubiquitous expression of POL in many plant tissues indicates that
POL may function to regulate signal transduction in multiple developmental
pathways. There is evidence to indicate that POL affects the activity of WUS,
a target of the CLV1 pathway. Although pol and wus are
normally both recessive mutations, wus is incompletely dominant in a
pol mutant background, indicating that POL and WUS
may function closely in the same pathway. Two models have been proposed for
the regulation of the CLV1 pathway by POL. One scenario consistent with
genetic analyses is that POL acts as a downstream negative regulator of CLV1
signaling. Alternatively, POL could function as an activator of CLV target
gene(s), and is itself inactivated by CLV signaling. More components of CLV
signaling will need to be isolated to resolve these questions of the mechanism
of POL function. POL and KAPP seem to act at different stages of the CLV1
signaling pathway, with KAPP thought to dephosphorylate the CLV1 receptor at
the plasma membrane and POL potentially dephosphorylating a downstream
intermediate of the signaling pathway.

Work on the CLV1 pathway is progressing rapidly in the identification of
signaling components, and in establishing the hierarchy of those components.
However, challenges remain, as many components have yet to be identified and,
more importantly, very few direct protein interactions have been assessed in
detail.

The BRI1 pathway

Steroid hormones are very important for physiological and developmental
regulation, both in animals and plants. In animals, steroid hormones are
recognized by ligand-dependent steroid nuclear receptors that promote the
transcription of specific target genes. The existence of receptors at the
plasma membrane involved in the rapid response to steroid hormone, also known
as non-genomic signaling, was postulated many years ago (reviewed by
Losel et al., 2003). However,
considerable controversy still remains over the nature or presence of
receptors that mediate the steroid response at the plasma membrane.

In A. thaliana, no homologs of steroid nuclear receptors are
known. However, steroid receptors at the plasma membrane have recently been
identified. Exogenous application of brassinosteroid (BR) to plants induces a
large range of phenotypes, including stem elongation, cell expansion of young
aerial tissues (such as hypocotyl and petioles), xylogenesis, leaf bending and
ethylene biosynthesis (Clouse,
2001). Plants unable to produce or perceive BR (see below) exhibit
phenotypes such as dwarfism, dark green leaves, reduced male fertility and
prolonged life span, and they respond inappropriately when grown in the dark
(Fig. 3A).

bri1 mutant plants and signaling. (A) Representation of wild type
and the severe dwarf phenotype observed in bri1 mutant plants.
Rosette leaves of bri1 plants are `cabbage-like', because of defects
in stem elongation, and cell expansion of hypocotyls and petioles. (B) Model
for the BRI1 signaling pathway. The type II serine carboxypeptide BRS1
activates a putative steroid binding protein (proSBP), which then associates
with the brassinosteriod BR. SBP-BR interacts with the LRR-RLK BRI1 (red) and
the receptor BAK1 (dark blue). BAK1/BRI1 then inactivate the GSK3-like kinase
BIN2, which is an upstream regulator of BES1 and BRZ1. In the absence of BR,
BIN2 is constitutively active, and phosphorylates BES1 and BRZ1, leading to
their degradation. When BES1 and BRZ1 are not phosphorylated, they are
localized to the nucleus where they activate transcription of brassinosteroid
responsive genes.

To identify components of the BR signaling pathway, genetic screens have
been performed to isolate mutants that are unable to respond to the exogenous
application of BRs, particularly the most bioactive compound, the steroid
hormone brassinolide (BL). The first mutant, brassinolide insensitive
1 (bri1), was identified in A. thaliana by its
BR-deficient phenotype and its failure to be rescued by exogenous BL treatment
(Clouse et al., 1996;
Li and Chory, 1997).
BRI1 is globally expressed in all plant tissues and its plasma
membrane localization made it a good candidate for a receptor that transduces
the BR signal across the plasma membrane in plants
(Friedrichsen et al., 2000).
Biochemical and genetic analyses have shown that both the extracellular domain
of the BRI1 receptor and, in particular, its 70 amino acid `island' located in
between the 21st and 22nd LRRs, are required for perception of BL. In the
first of these experiments, chimeric receptors were generated that contained
fusions of the extracellular domain of BRI1 and cytoplasmic signaling
sequences of the XA21 receptor, a rice LRR receptor kinase for disease
resistance. These chimeric receptors were able to activate defense response
genes when BL was applied exogenously to rice cell suspension culture
(He et al., 2000). This
revealed that the extracellular domain of BRI1 was responsible for its role in
perceiving BL. Interestingly, a mutation in the extracellular `island' domain
of the chimeric BRI1/XA21 receptor abolished this defense response,
demonstrating the importance of this domain for BL perception. In another
experiment, specific BL-binding activity was detected after BRI-GFP fusion
proteins from transgenic plants were immunoprecipitated using anti-GFP
antibodies (Wang et al.,
2001); this indicates that BRI1 is either the BL receptor, or a
rate-limiting component of the BL receptor complex.

Consistent with the BRI1-XA21 chimeric receptor studies indicating a
central role for the `island' domain in BL perception, only mutations in the
`island' domain greatly reduced the association of BRI1 with BL. Moreover, BL
association with BRI1 activates BRI1 phosphorylation in vivo and requires the
kinase activity of BRI1. Together, these results suggest that BRI1 is the
primary receptor for BR in A. thaliana. However, it has not yet been
shown that BRI1 is able to bind BR directly. Further studies will be needed to
determine whether another component, such as a steroid-binding protein, is
also required to mediate steroid binding to BRI1.

At least two putative steroid-binding proteins, which may be secreted, have
been identified in the A. thaliana genome
(Arabidopsis Genome Initiative,
2000; Li et al.,
2001a). Interestingly, a screen for suppressors of the weak,
partial loss-of-function bri1-5 allele has revealed that
overexpression of a type II serine carboxypeptidase, BRS1, can
suppress the bri1 phenotype (Li
et al., 2001a). This suppression is specific to the BRI1 receptor,
and depends on the presence of BR, on a functional BRI1 kinase domain and on
BRS1 protease activity. BRS1 is proposed to regulate an early event in BRI1
signaling, and putative steroid-binding proteins could be substrates of BRS1.
It is possible that if BRS1 is required to activate a BR-binding protein, then
elevated expression of BRS1 might increase the concentration of bound BR, thus
overcoming the reduce signaling capability of the bri1-5 isoform.

Last year, a role for another LRR-RLK, the BRI1-associated receptor kinase
(BAK1), was demonstrated by a genetic gain-of-function screen and biochemical
analyses (Li et al., 2002;
Nam and Li, 2002). BAK1 is a
member of the somatic embryogenesis receptor kinase (AtSERK) family, which
consists of five genes in A. thaliana. BAK1 (AtSERK3) possesses four
leucine zippers and five LRRs in its extracellular domain. Direct physical
interaction between BRI1 and BAK1 was observed both in a yeast two-hybrid
system and in vivo in A. thaliana
(Li et al., 2002;
Nam and Li, 2002). Although
BAK1 overexpression suppresses the phenotypes of weak bri1
alleles, the bak1 null phenotype is weaker than the bri1
null phenotype, indicating that BAK1 is partially dispensable for BRI1
signaling and/or that BAK1 homologous genes functionally overlap with
BAK1. BRI1 could potentially form heterodimers with other AtSERK family
members in the absence of BAK1. Two models have been proposed for BRI1/BAK1
activation (Fig. 3B). In the
first model, ligand-bound BRI1 activates BAK1, which then phosphorylates
downstream components to regulate gene expression
(Li et al., 2002). A second
model proposes that BRI1 and BAK1 form an inactive heterodimer, which is then
stabilized and activated by the binding of BR, allowing the
transphosphorylation of the kinase domains to activate downstream components
(Nam and Li, 2002). Whether
BAK1 binds to the brassinosteroid hormone itself is unclear. Further analyses
might investigate the mechanisms, sites and timing of phosphorylation of BRI1
and BAK1 in response to BR binding in vivo, and will help distinguish between
these hypotheses.

To date, no direct substrate for the BRI1/BAK1 complex has been described.
However, several proteins with roles downstream of the BRI1 receptor in the BR
signaling pathway have been uncovered. BRI1-EMS-SUPPRESSOR 1 (BES1) and
BRASSINAZOLE RESISTANT 1 (BRZ1) are two proteins that share 88% identity with
each other and are part of a protein family that has six known members in
A. thaliana (He et al.,
2002; Wang et al.,
2002; Yin et al.,
2002). Except for a putative nuclear localization signal in the
amino-terminal portion of both BES1 and BRZ1, these proteins do not possess
significant similarities to known proteins
(Zhao et al., 2002b). A
number of studies have sought to determine the roles of BES1 and BRZ1 in the
BRI1 signaling pathway. Semi-dominant and dominant mutations in the sequences
coding for the PEST domains of BES1 and BRZ1, respectively,
give rise to mutant plants that, in the dark, are resistant to the BR
biosynthesis inhibitor brassinazole. Although these mutant plants exhibit the
same phenotypes as one another in the dark, BES1 and BRZ1 seem to play
different roles in the light. When grown in the light, the gain-of-function
bes1-D mutants display constitutive BR response phenotypes which
include extensive elongation of leaves and stems. By contrast,
gain-of-function brz1-D mutants exhibit a semi-dwarf phenotype,
possibly due to the activation of a feedback mechanism that inhibits the
normal BR signaling pathway.

BES1 and BRZ1 activity may be regulated at the protein level
(Fig. 3B)
(He et al., 2002;
Wang et al., 2002;
Yin et al., 2002;
Zhao et al., 2002b). BL
treatment increases both the accumulation of, and the nuclear localization of,
unphosphorylated BES1 and BRZ1 proteins. This effect is dependent on BRI1,
which indicates that BES1 and BRZ1 are downstream specific, positive
regulators of the BR signaling pathway. One could infer from these results
that, in the absence of BL, a negatively acting kinase phosphorylates BES1 and
BRZ1, leading to their degradation. Several studies indicate that this
upstream repressor of BES1 and BRZ1 is the ser/thr kinase
BRASSINOSTEROID-INSENSITIVE 2 (BIN2) (Li
and Nam, 2002; Li et al.,
2001b). Gain-of-function mutations of this GSK3/SHAGGY-like kinase
result in plants that exhibit a BR-deficient phenotype suppressed by
bes1-D, suggesting that BIN2 is likely to function upstream of BES1
(Yin et al., 2002). In
addition, BRZ1 and BES1 both possess multiple consensus sequences (S/TxxxS/T)
known to be phosphorylated by GSK3/Shaggy kinases. Finally, BIN2 interacts in
yeast cells with BES1 and BRZ1, and phosphorylates both proteins in vitro.
These data indicate that in the absence of BR, constitutively activated BIN2
phosphorylates BES1 and BRZ1, which is likely to lead to the degradation of
phosphorylated BES1 and BRZ1 proteins (Fig.
3B). In the presence of BR, activation of the BRI1 complex at the
plasma membrane leads to the inhibition of the BIN2 kinase activity. This, in
turn, permits the accumulation of unphosphorylated BES1 and BRZ1 proteins that
can translocate to the nucleus to activate their specific BR responsive
targets. In agreement with the bes1 and brz1 mutant
phenotypes, microarray analyses show that BES1 activates the transcription of
genes that encode the cell-wall modifying enzymes required for cell
elongation, and BRZ1 activates the transcription of genes involved in the BR
negative-feedback pathway that inhibits BR-induced cell elongation in a
light-dependent manner (Wang et al.,
2002; Yin et al.,
2002).

The model proposed for the BR pathway activation by BES1 and BRZ1 is
reminiscent of the mechanism described for the Wingless/Wnt signaling pathway.
In models of both systems, the signal (Wnt in animals versus BR in plants)
activates a plasma-membrane receptor (Frizzled versus BR1 and BAK1) that
activates signaling by inactivating a GSK3-like kinase (Shaggy versus BIN2),
which normally functions to repress, through phosphorylation and degradation,
downstream effector(s) (β-catenin versus BES1 and BZR1) (reviewed by
Cadigan and Nusse, 1997).
Clearly remarkable progress has been made on both identifying the components
of BRI1 signaling, as well as some of the key protein interactions. Pressing
questions include understanding the nature of BL interaction with BRI1 (e.g.,
is it mediated by a BL-binding protein?), and the mechanism by which BRI1 and
BAK1 regulate BIN2 activity.

LRR-containing receptors and defense responses in plants and
animals

The Drosophila melanogaster Toll receptor and its mammalian
homologs, the Toll-like receptors (TLR), have extracellular LRR domains
structurally similar to many plant RLKs. However, Toll differs from plant RLKs
in that its cytoplasmic domain is composed of about 200 amino acids called the
Toll/interleukin 1 receptor (TIR) domain
(Hashimoto et al., 1988;
Takeda et al., 2003). The
Toll pathway was first identified in D. melanogaster on the
basis of its role in dorsoventral patterning during early embryogenesis
(Hashimoto et al., 1988).
Genetic and molecular analyses are consistent with a model in which a
proteolytic cascade activated on the ventral side of the pre-cellular embryo
processes the secreted cytokine Spätzle, a member of the cysteine knot
family of growth factors, which activates the Toll receptor
(Lemaitre et al., 1996;
Weber et al., 2003). Toll
activation recruits a complex containing at least three proteins
(Sun et al., 2002), the
adaptor MyD88/Krapfen (Kra) (Charatsi et
al., 2003), the scaffolding Tube protein
(Letsou et al., 1991;
Letsou et al., 1993), and the
cytoplasmic ser/thr protein kinase Pelle
(Hecht and Anderson, 1993).
Interestingly, the kinase domain of Pelle is the animal protein kinase most
similar to the cytoplasmic ser/thr kinase domains of plant LRR-RLKs
(Shiu and Bleecker, 2001b).
Several studies have demonstrated that Kra binds to Toll by their respective
TIR domains, and that Kra, Tube and Pelle form an heterotrimeric complex bound
by their death domains, which are widely involved in protein-protein
interactions (Sun et al.,
2002). This aggregation triggers phosphorylation and degradation
of the IκB-like inhibitor Cactus, so that the Rel/NFκB
transcription factor Dorsal is released and translocates to the nucleus
(Galindo et al., 1995;
Grosshans et al., 1994). In
the nucleus, Dorsal directs the expression of a number of downstream factors
(Thisse et al., 1991).

In adult D. melanogaster, the Toll pathway is also involved in
innate immunity (Lemaitre et al.,
1996). The mechanism of intracellular signaling that occurs upon
fungal and bacterial infection is essentially the same as that described above
for dorsoventral axis formation, with the exception that Spätzle is
generated in response to fungal products through the activation of the serine
protease Persephone (Ligoxygakis et al.,
2002), and that the Rel factor translocated in the nucleus is the
Dorsal-related immunity factor (Dif), which upregulates the transcription of
antimicrobial genes (Ip et al.,
1993). In humans, ten Toll-like receptors are implicated in the
recognition of pathogen-associated molecular patterns (PAMPs)
(Dunne and O'Neill, 2003). The
ligands identified include bacterial lipopeptides, peptidoglycan, dsRNA,
fibronectin and flagellin. These ligands possess the same functional domains
as the ligands of their Toll homolog in D. melanogaster, and all
Toll-like receptors use TIR domain-containing adaptors to transduce the signal
through the activation of two Interleukin-1 receptor-associated kinases
(IRAKs), homologs of the Pelle ser/thr kinase cytoplasmic protein in the fly
(Cao et al., 1996a). IRAKs
phosphorylate and activate the tumor necrosis factor (TNF) and the
receptor-associated factor 6 (TRAF6) (Cao
et al., 1996b). Activated TRAF6, in turn, activates the
mitogen-activated protein kinases (MAPK) and the mitogen-activaged protein
kinase kinase kinase (MAPKKK), called transforming growth factorβ
-activated kinase (TAK1). TAK1 can phosphorylate the IκBα
kinase complex, as well as MKK (which are MAPKKs), leading to activation of
the p38 MAPKs and c-Jun N-terminal kinase (JNK)
(Takaesu et al., 2000).

Interestingly, the animal developmental/defense response pathways described
above have some parallels with certain signaling pathways in plants. Both
plants and animals use LRR-containing receptors to detect specific pathogenic
peptides, both use Pelle-family kinases to relay signals from the receptors,
and both use MAPK pathway kinases to activate downstream defense responses. A
screen for mutants insensitive to flagellin (major antigens of several
bacteria) upon bacterial infection led to the discovery of the FLS2
locus in A. thaliana (Gomez-Gomez
et al., 1999). The FLS2 gene codes for an LRR-RLK
(Gomez-Gomez and Boller,
2000). Mutations in both the LRR and the kinase domains of FLS2
affect the binding activity of the most conserved domain of flagellin, the
peptide flg22 (Gomez-Gomez et al.,
2001). This binding activity is rescued when a wild-type FLS2
receptor is expressed in the fls2 mutant plants, indicating that FLS2
is the receptor for the flg22 peptide, and that the kinase activity of the
FLS2 receptor is necessary for the binding of flg22. The kinase-associated
protein phosphatase KAPP interacts with the kinase domain of FLS2 in a yeast
two-hybrid assay, and plants overexpressing KAPP are insensitive to flagellin
treatment (Gomez-Gomez et al.,
1999). These data indicate that KAPP is a negative regulator of
the FLS2 signaling pathway. New data have shown that FLS2 directly or
indirectly activates a cascade of phosphorylation, implicating the MAPK
pathway proteins MEKK1, MKK4/MKK5 and MPK3/MPK6 in this signaling pathway
(Asai et al., 2002). The
targets of this signal include the well-described defense genes, such as
PAL1, GST1, PR1 and PR5, induced in many plant species, as
well as the WRKY22/WRKY29 transcription factors and LRR-RLK FRK1
(FLG22-induced receptor-like kinase 1). Interestingly, the activation of the
MAPK cascade confers resistance to bacteria and fungi, indicating a
convergence of the signal induced by different pathogens to this specific
cascade. Moreover, the results also indicate the presence of a
MAPK-independent pathway, which could be calcium-dependent, for the activation
of other defense-responsive factors.

Conclusions

One important feature of signal transduction in animals is that receptors
are usually the starting point of a complex array of signaling pathways, where
cross-talk, feedback loops, branch points and multi-component signaling
complexes converge to enable the transcription of target genes in the nucleus.
There is no reason to think that this feature is not also true in plants.
Thus, for the three pathways described in this review, the CLV1, BRI1 and FLS2
pathways, the simple linear models of their function are likely to represent,
at best, only a portion of very complex signaling circuitries. Already for the
CLV1 pathway, characterization of the signaling regulator POL has revealed a
WUS-independent CLV1 pathway regulating meristem development
(Yu et al., 2003;
Yu et al., 2000).

The RLK pathways may be further complicated by functional redundancy
between receptors. There are 216 LRRRLKs in the A. thaliana genome,
but only a handful have been associated with a biological function. One
possible reason that the function of so many receptors is unknown is that
multiple receptors may functionally overlap with one another, such that mutant
versions of the receptors would not show up in straightforward genetic
screens. Indeed, receptors with functional overlap are strongly implicated in
both the CLV1 and ERECTA pathways (Table
1) (Diévart et al.,
2003; Shpak et al.,
2003). Additional functional redundancy has been detected at the
level of ligand function. The putative CLV1 ligand CLV3 is part of a family
that contains at least 20 CLV3-like (CLE) members
(Sharma et al., 2003). CLE
members are expressed in several tissues during development, and some of them,
such as CLV3, are secreted into the periplast. One of them, CLE40, when
ectopically expressed in the meristem of transgenic plants, is functionally
equivalent to CLV3 (Hobe et al.,
2003). However, in wild-type plants, its level of expression is
ubiquitous and low, and the loss of CLE40 in A. thaliana
enhances root waving. Thus, a CLV3-like protein plays a role in root
development, indicating that a CLV1-like receptor may receive and transduce
the CLE40 signal in roots.

Studies on gene families that encode ligands, receptors and signaling
components are likely to yield much new information on the diversity and
specificity of pathways downstream LRR-RLKs in plants. Open questions still
remain as to the function of more than 200 LRR-RLKs that are as yet without an
attributed signaling mechanism. We know little about whether mechanisms of
signaling identified to date for receptors such as BRI1, FLS2 and CLV1 are
common among those receptors that remain unknown. We might hope, however, that
many receptors will use common downstream components and mechanisms to relay
signals from the plasma membrane to the nucleus. As we have described, the
protein phosphatase KAPP appears to play a role in negatively regulating the
signaling of many receptors. Moreover, the gene encoding the protein
phosphatase POL, which regulates CLV1 signaling, is expressed in many tissues
in A. thaliana, exhibits mutant phenotypes outside of the meristem,
and has sequence similarity with several other POL-like genes,
indicating that the function of this family of PP2C could be conserved
downstream of multiple LRRRLKs (Yu et
al., 2003; Yu et al.,
2000). A close BRI1 homolog, VH1, appears to regulate vascular
development in A. thaliana (Clay
and Nelson, 2002). One wonders whether VH1 regulates vascular
development through BIN2 or a BIN2-related protein. Will other pathogen
responses use similar signaling mechanisms to those that are being uncovered
for FLS2? These and related questions will certainly occupy researchers for
years to come.

Hecht, P. M. and Anderson, K. V. (1993).
Genetic characterization of tube and pelle, genes required for signaling
between Toll and dorsal in the specification of the dorsal-ventral pattern of
the Drosophila embryo. Genetics135,405
-417.

Lenhard, M. and Laux, T. (2003). Stem cell
homeostasis in the Arabidopsis shoot meristem is regulated by intercellular
movement of CLAVATA3 and its sequestration by CLAVATA1.
Development130,3163
-3173.

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